Plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus, chimeric derivatives thereof, and method of producing an infectious bovine viral diarrhea virus using said plasmid

Abstract

A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a vector in many advantageous applications.

The following is claimed: 
     
       1. A method of producing recombinantly engineered infectious bovine viral diarrhea virus comprising: providing a vector comprising a reverse transcribed copy of bovine viral diarrhea virus RNA;   recombinantly engineering the reverse transcribed copy of the bovine viral diarrhea virus;   transforming an E. coli with the vector;   causing the transformed host cell to produce a plurality of vectors;   extracting the plurality of vectors from the transformed host cell;   forming a plurality of synthesized RNA from the plurality of vectors comprising the reverse transcribed copy of the bovine viral diarrhea virus RNA;   introducing the synthesized RNA into a mammalian cell; and   recovering the recombinantly engineered infectious bovine viral diarrhea virus from the mammalian cell.   
     
     
       2. The method of claim 1 wherein the E. coli is strain GM119. 
     
     
       3. The method of claim 1 wherein the step of introducing the bovine viral diarrhea virus RNA into the mammalian cell utilizes electroporation. 
     
     
       4. The method of claim 1 wherein the mammalian cell is an embryonic bovine tracheal cell. 
     
     
       5. The method of claim 1 wherein the vector comprises pVVNADL-SINgp. 
     
     
       6. The method of claim 1 wherein the vector contains a selective marker for transformed cells. 
     
     
       7. The method of claim 1 wherein the vector comprises pVVNADLΔDra. 
     
     
       8. A method of producing recombinantly engineered infectious bovine viral diarrhea virus comprising: providing a vector comprising a reverse transcribed copy of bovine viral diarrhea virus RNA;   recombinantly engineering the reverse transcribed copy of the bovine viral diarrhea virus to form a recombinantly engineered vector;   transforming E. coli, strain GM119, with the recombinantly engineered vector;   causing the transformed E. coli to produce a plurality of the recombinantly engineered vectors;   extracting the plurality of recombinantly engineered vectors from the transformed E. coli;   forming a plurality of synthesized RNA from the plurality of recombinantly engineered vectors comprising the reverse transcribed copy of bovine viral diarrhea virus RNA;   introducing the synthesized RNA into an embryonic bovine tracheal cell by electroporation; and   recovering the recombinantly engineered infectious bovine viral diarrhea virus.   
     
     
       9. A method of isolating a plurality of recombinantly engineered bovine viral diarrhea virus comprising: providing a vector comprising a reverse transcribed copy of a genome of bovine viral diarrhea virus RNA;   creating a recombinantly engineered infectious bovine viral diarrhea virus;   causing a host cell to produce a plurality of recombinantly engineered infectious bovine viral diarrhea virus; and   isolating the plurality of recombinantly engineered bovine viral diarrhea virus.   
     
     
       10. A method of producing recombinantly engineered infectious bovine viral diarrhea virus comprising: providing a pVVNADL;   recombinantly engineering pVVNADL to form a recombinantly engineered pVVNADL;   transforming E. coli, strain GM119, with the recombinantly engineered pVVNADL to form a transformed E. coli;   causing the transformed E. coli to produce a plurality of the recombinantly engineered pVVNADL;   extracting the plurality of recombinantly engineered pVVNADL from the transformed E. coli;   forming a plurality of synthesized RNA from the plurality of recombinantly engineered pVVNADL;   introducing the plurality of synthesized RNA into an embryonic bovine tracheal cell by electroporation; and   recovering the recombinantly engineered infectious bovine viral diarrhea virus.   
     
     
       11. An isolated vector adapted to produce recombinantly engineered infectious bovine viral diarrhea virus. 
     
     
       12. The vector of claim 11, further comprising a sequence tag. 
     
     
       13. A chimeric vector comprising a 270 nucleotide insert and an envelope glycoprotein from Singer strain bovine viral diarrhea virus wherein the vector is adapted to produce a chimeric infectious bovine viral diarrhea virus. 
     
     
       14. The vector of claim 13 wherein the vector comprises pVVNADL-SINgp. 
     
     
       15. The vector of claim 13, wherein the synthesized RNA is introduced into EBTr cells. 
     
     
       16. An isolated vector comprising sequence ID. NO. 1. 
     
     
       17. A vector comprising pVVNADL-SINgp. 
     
     
       18. A vector comprising pVVNADLΔDra. 
     
     
       19. A chimeric virus comprising i-VVNADL-SINgp. 
     
     
       20. A recombinant bovine viral diarrhea virus comprising i-VVNADLΔDra. 
     
     
       21. A plurality of recombinantly engineered infectious bovine viral diarrhea virus produced by the process comprising: providing a vector comprising a reverse transcribed copy of a genome of bovine viral diarrhea virus RNA;   creating a recombinantly engineered infectious bovine viral diarrhea virus;   causing a host cell to produce a plurality of recombinantly engineered infectious bovine viral diarrhea virus; and   isolating the plurality of recombinantly engineered infectious bovine viral diarrhea virus.